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W Wu C Xu X Ling C Fan B P Buckley M V Chernov L Ellis F Li I G Mu?oz X Wang 《Cell death & disease》2015,6(12):e2035
Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2–p53 interface or MdmX ((MDM4), mouse double minute 4)–p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2–MdmX really interesting new gene (RING)–RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2–MdmX RING–RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2–MdmX RING domain inhibitors)) that specifically inhibit Mdm2–MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2–MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2–MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.Activation of tumor-suppressor p53 as a targeted non-genotoxic cancer therapy has been pursued for many years,1, 2 because p53 possesses potent tumor-suppressing activity in vivo.3, 4, 5 p53 can inhibit cancer cell growth by cell cycle arrest or terminate their proliferation by inducing apoptosis and senescence.6 The p53-based therapy is particularly attractive for cancer types including retinoblastoma, neuroblastoma and leukemia/lymphoma in which p53 is rarely mutated7 and p53-dependent apoptotic pathway is a predominant endpoint.8, 9, 10 Except for cancer-selected p53 mutations, the p53 activity is mainly inhibited by p53-binding proteins Mdm2 and MdmX ((MDM4), mouse double minute 4) in normal and cancer cells.11, 12 Prior focus of p53 reactivation strategy has been on targeting the Mdm2–p53 and/or MdmX–p53 interface. This has led to the discovery of a list of potent Mdm2–p53 inhibitors13 with several compounds of this class being advanced to phase I clinical trials in hematological neoplasia and solid tumors.2 However, the therapeutic effects of these Mdm2–p53 inhibitors can be attenuated by MdmX overexpression.14, 15, 16 Although peptide inhibitors with dual functions of inhibiting both Mdm2–p53 and MdmX–p53 interactions will overcome this problem and enhance p53-dependent cancer killing;17, 18 these inhibitors will not inhibit Mdm2 E3 ligase activity toward non-p53 targets such as retinoblastoma protein (RB), p21 and DAXX (death domain-associated protein),19, 20, 21 which to a different extent contributes to the p53-dependent biological effects.Recent genetic studies indicated that really interesting new gene (RING) domains of Mdm2 and MdmX are required for in vivo inhibition of p53 activity during development.22, 23, 24 MdmX was reported to stimulate Mdm2-mediated p53 multiple monoubiquitination using glutathione S-transferase (GST) fusion Hdm2 proteins.25, 26 Using non-GST Hdm2 proteins in in vitro biochemical assays, we found that MdmX–Mdm2 RING–RING interaction is essential for p53 polyubiquitination and proteasome-dependent degradation.26 These findings established that Mdm2–MdmX complex is the key regulator of p53 activity and Mdm2–MdmX RING–RING interaction is a critical but an unexplored interface for drug targeting.27 Identification of E3 ligase inhibitors for cancer therapy presents a huge opportunity but with great challenges.28 In this report, we describe successful identification and characterization of small molecule inhibitors for the E3 ligase activity of Mdm2–MdmX E3 complex. Among seven specific MMRis (Mdm2–MdmX RING domain inhibitors), MMRi64 was followed up in detail in this report. MMRi64 has several unique features that distinguish it from Mdm2–p53 inhibitor Nutlin3a. MMRi64 disrupts Mdm2–MdmX interaction in vitro and inhibits the E3 ligase activity of Mdm2–MdmX without affecting the E3 ligase activity of Mdm2 RING domain homodimers. MMRi64 induces p53 accumulation without induction of Mdm2 and p21 in lymphoma cells, which is distinct from the effects of Nutlin3a. Finally, MMRi64 induces PUMA (p53 upregulated modulator of apoptosis) but strongly downregulates MdmX and Mdm2, consequently activating the apoptotic arm of the p53 pathway in leukemia/lymphoma cells without the induction of growth arrest. 相似文献
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O Fortunato M Boeri M Moro C Verri M Mensah D Conte L Caleca L Roz U Pastorino G Sozzi 《Cell death & disease》2014,5(12):e1564
Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21WAF1/CIP1 in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.Lung cancer is the leading cause of cancer death worldwide, resulting in >1.4 million deaths/year.1 Lung tumors are often discovered as locally advanced or metastatic disease, and despite improvements in molecular diagnosis and targeted therapies, the overall 5-year survival rate remains in the 10–20% range. Indeed, nonsmall cell lung cancer (NSCLC) is poorly chemosensitive to most of the available agents with response rates ranging from 10 to 25%.2 The discovery of recurrent mutations in the epidermal growth factor receptor (EGFR) kinase,3 as well as gene fusion products involving the anaplastic lymphoma kinase (ALK),4 has led to a marked change in the treatment of patients with lung adenocarcinoma, the most common type of lung cancer.5, 6 To date, patients with mutations in the EGFR gene, suitable for targeting by EGFR tyrosine kinase inhibitors, represent roughly 10%, whereas the subgroup of tumors with ALK rearrangements, targeted by ALK inhibitors, is only ~5%.7 Thus, the majority of lung tumors lack effective treatment and novel therapeutic strategies are still needed.MicroRNAs (miRNAs) are short noncoding RNAs, 20–24 nucleotides long, that have important roles in almost all biological pathways,8, 9, 10, 11 and influence cancer-relevant processes, such as proliferation,12 cell cycle,13 apoptosis,14 and migration.15 Many studies have reported the critical role of miRNAs in lung cancer pathogenesis and their potential as biomarkers for lung cancer risk stratification,16 outcome prediction,17 and classification of histological subtypes.18, 19 miRNAs are actively released by various cell types and can be detected in biological fluids, such as plasma and serum, making them suitable as circulating biomarkers in NSCLC.20, 21There is limited evidence of mir-660 deregulation in cancer and little is known about its role in lung tumorigenesis and its putative target genes. Mir-660 has been reported to be upregulated in chronic lymphocytic leukemia22, 23 and in leukemic cells after treatment with 4-hydroxynonenal, a compound that induces differentiation and blocks proliferation of leukemic cells.24 In a previous study we demonstrated that mir-660 was one of the 24 miRNAs deregulated in plasma samples of NSCLC patients identified in a low-dose computed tomography (LDCT) screening trial.20The p53 tumor suppressor protein is a key regulator of cell cycle G0/G1 checkpoint, senescence, and apoptosis in response to cellular stress signals.25, 26 Mouse double minute 2 (MDM2), a p53–E3 ubiquitin ligase,27 is the principal negative regulator of the expression level and function of p53.28, 29 Several studies have illustrated different mechanisms of p53 regulation by MDM2,30, 31 such as binding transactivation region of p53,32, 33 promoting nuclear export and cytoplasmic accumulation of p53 by monoubiquitination,34, 35 and inducing p53 proteosomal degradation by polyubiquitination.36 In addition, MDM2 gene has been reported to be amplified or overexpressed in a variety of human cancers, such as sarcoma,37 lymphoma,38 breast cancer,39 lung cancer,40 and testicular germ cell tumor.41 Several miRNAs targeting MDM2 have been identified, such as the mir-143/mir-145 cluster that can be induced by p53,42 as well as mir-25 and mir-32, known to inhibit tumor glioblastoma growth in mouse brain.43In this study, we report that mir-660 is downregulated in tissue and plasma samples of lung cancer patients and demonstrate that mir-660 replacement impairs the functionality of p53 wild-type (wt) lung cancer cells and inhibits in vitro and in vivo tumor growth. We showed that all the effects observed after mir-660 overexpression were absent in p53 ko cells, identified MDM2 as mir-660 direct target gene and indicate impairment of the MDM2/p53 interaction as the mechanism underlying tumor growth inhibition. 相似文献
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Y Shetzer S Kagan G Koifman R Sarig I Kogan-Sakin M Charni T Kaufman M Zapatka A Molchadsky N Rivlin N Dinowitz S Levin G Landan I Goldstein N Goldfinger D Pe'er B Radlwimmer P Lichter V Rotter R Aloni-Grinstein 《Cell death and differentiation》2014,21(9):1419-1431
p53 loss of heterozygosity (p53LOH) is frequently observed in Li-Fraumeni syndrome (LFS) patients who carry a mutant (Mut) p53 germ-line mutation. Here, we focused on elucidating the link between p53LOH and tumor development in stem cells (SCs). Although adult mesenchymal stem cells (MSCs) robustly underwent p53LOH, p53LOH in induced embryonic pluripotent stem cells (iPSCs) was significantly attenuated. Only SCs that underwent p53LOH induced malignant tumors in mice. These results may explain why LFS patients develop normally, yet acquire tumors in adulthood. Surprisingly, an analysis of single-cell sub-clones of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that p53LOH is a bi-directional process, which may result in either the loss of wild-type (WT) or Mut p53 allele. Interestingly, most BM progenitors underwent Mutp53LOH. Our results suggest that the bi-directional p53LOH process may function as a cell-fate checkpoint. The loss of Mutp53 may be regarded as a DNA repair event leading to genome stability. Indeed, gene expression analysis of the p53LOH process revealed upregulation of a specific chromatin remodeler and a burst of DNA repair genes. However, in the case of loss of WTp53, cells are endowed with uncontrolled growth that promotes cancer.Heterozygosity, caused by a mutation in a single allele of a tumor suppressor gene (TSG), is one of the first steps in malignant transformation.1 Often, TSGs undergo loss of the wild-type (WT) allele, designated as loss of heterozygosity (LOH).2, 3, 4 Patients with the rare cancer predisposition Li-Fraumeni syndrome (LFS), carrying germ-line heterozygous p53 mutations,5 apparently exhibit normal development yet later in adult life develop a wide spectrum of tumors; predominantly sarcomas,6, 7, 8 where 40–60% of tumors exhibit WT p53 loss of heterozygosity (p53LOH).8Giving that cancer development could be associated with stemness deregulation challenges, the notion that the occurrence of p53LOH in stem cells (SCs) may contribute to the emergence of cancer SCs. Genomic fidelity is a hallmark of SCs.9 The genome of embryonic stem cells (ESCs) is extremely stable, whereas adult stem cells (ASCs) exhibit a less stable genome.10 Genetic deregulation in ASCs was shown to be associated with tumor development.11, 12, 13 Mesenchymal stem cells (MSCs) that acquire mutations in oncogenes/TSGs such as p53 may function as tumor-initiating cells leading to de-novo sarcomagensis.14, 15, 16, 17 Furthermore, MSCs isolated from young mice, aged in culture acquired clinically relevant p53 mutations.18 In all, these findings suggest a link between p53 inactivation in SCs and tumorigenesis.Although induced pluripotent stem cells (iPSCs) seemed to represent ESCs,19, 20 several studies questioned the assumption that iPSCs are as genomically stable as ESCs.21, 22, 23, 24 p53 was found to have a major role in the generation of iPSCs both in attenuating reprogramming and controlling the quality of the reprogrammed cells.25, 26 An additional role of p53 during reprogramming may be an indirect effect on cell proliferation27 and on the restriction of mesenchymal–epithelial transition during the early phases of reprogramming.28 Importantly, Mutp53 cells exhibiting a fully reprogrammed iPSC phenotype in vitro, form malignant tumors in vivo, instead of the benign teratomas induced by the WTp53-iPSCs.25 As p53 is the guardian of the genome, it is important to investigate how p53LOH would affect genome stability and tumorigenicity of iPSCs.The availability of in vitro SC p53LOH models (iPSCs, MSCs) can help decipher the role of p53LOH in cancer initiation. Indeed, the incidence of p53LOH was found to be extremely different between these SCs. Surprisingly, we found that reprograming of heterozygous p53 (HZp53) fibroblasts, which frequently undergo p53LOH, gave rise to iPSC clones, most of which retained their HZp53 status and exhibited features of normal WTp53-iPSCs. However, p53LOH process is robust in MSCs. Interestingly, single-cell sub-cloning of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that, in addition to the loss of the WTp53, loss of the Mutp53 allele also takes place. Of note, this bi-directional p53LOH occurred in an age-dependent manner linking LOH to aging and tumorigenesis. Surprisingly, most of the p53LOH events in BM progenitors preferred the loss of the Mutp53 allele. Taken together, our results of a bi-directional p53LOH process, accompanied by a burst of DNA repair pathways, may suggest that p53LOH can be regarded as a DNA repair event. In the case of a DNA repair-orientated productive LOH process, where the Mutp53 allele is lost, cells are rescued of tumorigenesis. However, when the WTp53 allele is lost, cells become prone to tumor initiation. 相似文献
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Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.1 Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.2, 3, 4 Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.2, 5, 6, 7 Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.8, 9, 10, 11, 12 Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.8, 11, 12 Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.8, 9 Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,13 liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.12, 14, 15Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon16, 17 and prostate cancers,18, 19 contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis20 and is necessary for tBid-induced cancer cell apoptosis.21 Loss of Bax has been reported to promote tumor development in animal models.22 Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.23, 24, 25 p53 has been reported to be a tumor suppressor,26 and its mutant can cause chemoresistance in cancer cells.27, 28, 29 Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.30, 31 Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.32 Some studies have revealed that Bak is a determinant of cancer cell apoptosis.33, 34 Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.33, 35 In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells. 相似文献